Cryopreservation, particularly the cryopreservation of biological samples, refers to the freezing/vitrification of tissues or cells in order to preserve them for future use. Unfortunately, as recognized by those of ordinary skill in the art, cryopreservation and thawing, of biological samples using presently available techniques often is less than optimal, as the biological activity of certain biological samples is often significantly diminished as a result. The practical use of particular biological techniques dictates certain cells are stored for long periods of time. There has been a growing demand for a new method of preserving biological samples, in particular cellular material, such that the biological functionality of the material is preserved after warming. A particular need for the preservation of biological samples has arisen in nuclear transfer and in vitro embryo production.
Cryopreservation provides an instrumental step in the embryo technology field, in particular relating to bovine because embryos can be stored until recipients are synchronized. However, previous cryopreservation techniques either fail to preserve high embryo viability or fail to provide an efficient means for cryopreserving embryos, particularly for bovine embryos produced in vitro.
Conventionally, biological samples were preserved by either slow freezing or by vitrification. The slow freezing technique involves lowering the temperature of a chamber in a stepwise manner. This technique has been used successfully in mouse, human and bovine embryos produced in vivo. However, this process does not work well with in vitro fertilized embryos, and particularly doesn't work well with bovine IVF embryos, possibly due to the different compositions and distributions of proteins and lipids as compared to embryos produced in vivo. Specifically, bovine IVF embryos tend to have high concentrations of lipids because of the cultures in which they are developed. These lipids can result in an embryo which is sensitive to chilling/freezing presumably because the lipids interfere with the intracellular/extracellular exchange of water required for slow freezing. Water and other fluids must be removed during this process or potentially toxic cryopreservatives must be added to prevent intracellular fluids from freezing during the slow freezing process. Otherwise, fluids, such as water, crystallize in a lattice structure which expands from the liquid volume. This expansion causes intracellular stress and mechanical damage to the developing embryonic cellular mass, and can be damaging enough to affect an embryo's viability. The addition of toxic cryoprotectants and attempts to dehydrate IVF embryos mitigate this damage but have yielded less than satisfactory results.
Vitrification provides an alternative means for preservation. Vitrification can be defined as the solidification of a solution brought about not by crystallization, but by extreme elevation in viscosity during cooling. In the case of embryos vitrification has been achieved by freezing the entire embryo so quickly that water molecules do not have time to adjust into a crystallized lattice structure. Instead, the mater molecules remain in their random configuration. By retaining the same ionic and molecular distribution in a glass state chemical and mechanical damage to adjacent intracellular components is avoided.
It is believed that one of the bottlenecks of vitrification technology is the “insufficient” cooling rate of oocytes in current vitrification schemes (Vajta et al., Embryo Transfer Newsletter 15: 12-18 (1997)). In order to overcome this problem, several methods have been proposed, which use very small amounts of solution. However, a need still exists for a practical technique for preserving embryos with excellent cooling rates.
So-called “minimum drop vitrification” systems have allowed breakthrough results with bovine and porcine oocyte cryopreservation (See, e.g., Arav A., Vitrification of oocyte and embryos, In: Lauria A, Gandolfi F (eds.), New trends in embryo transfer, Cambridge, England: Portland Press, 255-264 (1992)). In “minimum drop vitrification” small amounts of solution are placed on a special cryo-stage which is cooled down quickly. This method, unfortunately, has not been found by the art to be convenient for preserving large numbers of oocytes.
Another vitrification technique includes loading a few microliters of vitrification solution into glass capillaries (Dinnyes et al., Cryobiology 31: 569-570 (1994)), or into open pulled plastic straws (Vajta et al., Mol. Reprod. Dev. 51: 53-58 (1998)) and plunging the capillary or straw quickly into liquid N2. Similarly, vitrification success was achieved by plunging oocyte-containing vitrification solutions with a small loop (Lane et al., Theriogenology 51: 167 (1999) (abstr.)). However, such techniques have not been found highly efficient presumably because plunging a warm object into liquid N2 results in the boiling of the liquid and for a short time creates an isolating layer of N2 vapor around the object.
In order to reduce the possibility of an isolating layer of vapor interfering with efficient vitrification, it has been proposed that oocyte-containing vitrification solution be dropped directly into liquid N2. Such technique has been reported to be slightly more effective than prior art vitrification techniques. (Riha et al., Zivoc. Vir. 36: 113-120 (1991); Papis et al., Theriogenology 51: 173 (1999) (abstr.); Yang et al., Theriogenology 51: 178 (1999) (abstr.)), presumably by eliminating the insulation effect of the vapor. However, such technique suffers from the problem of vitrified oocyte retrieval. Specifically, the vitrified oocytes, which can barely be perceivable under normal circumstances, are generally transparent and can be nearly impossible to retrieve in even a few millimeters of liquid. Some groups reported improved success of the cryopreservation of biological materials by using metal surfaces cooled down with the aid of liquid N2. Such metal surfaces are asserted to provide a more efficient heat transfer and to increase further the cooling rates than the cryo-stages used in minimum drop vitrification. Drosophila embryos were successfully preserved by placing them in a metal grid on a cold metal surface (Steponkus et al., Nature 345: 170-172 (1990)). Again, presently available techniques employing cooled metal surfaces have not been found convenient for preserving large numbers of oocytes.
U.S. Pat. No. 6,982,172, which is incorporated herein by reference, describes a method for Solid Surface Vitrification (SSV) of oocytes. The patent describes a method including the steps of suspending the oocyte in an equilibrium fluid, rinsing the oocyte in a vitrification medium, and dropping the medium directly on a solid surface cooled to about −150° Celsius. The step of suspending an oocyte in an equilibrium fluid takes 12-15 minutes as described in this application. Once vitrified, the described technique provides for a single cooling surface, generally limited to about −150° Celsius that creates difficult to manipulate flattened vitrified “pies”. Therefore, a need exists for a technique with improved cooling rates not limited by the temperature of a solid surface and a further need exists for an improved cooling rate and an improved system for retrieving vitrified materials.
A further need exists for overcoming the problems of vitrified oocyte retrieval by direct dropping method mentioned above. Still a further need exists for a shortened incubation period and a more efficient method to vitrify a large number of embryos and oocytes. Finally, a need exist to vitrify biological material in a form easier to manipulate compared to flattened pies on a solid surface.
Yet still a further a need exists for a more efficient method to vitrify biological samples, and in particular a faster means to accommodate vitrifying a large number of embryos, which does not suffer drawbacks or difficulty in retrieval.
A need also exists for an improved method of vitrifying sexed embryos, or embryos created with sexed sperm, nuclear transfer embryos, or embryos generated by parthenogenetic activation. Additionally, a need exists for an improved method of vitrifying oocytes, whether oocytes are derived from aspraiation from slaughterhouse ovary, or by ovum pick-up oocyte retrieving from live animals, or derived by stem cells, or induced pluripotent stem (iPS) cells, or by other means.